Antifungal Activity of Plantago lanceolata and Sida ovata Leaf Extracts against Dermatomycotic Fungi

Plantago lanceolata and Sida ovata have been used as medicinal plants for centuries to cure numerous diseases. This study aimed to evaluate antifungal activity of P. lanceolata and S. ovata leaf extracts against dermatomycotic fungi. Crude extracts from leaves of both plants were prepared using methanol and ethyl acetate. Phytochemical screening of both plants leaves was performed. Antifungal activity of crude extracts was evaluated against three dermatomycotic fungi (Candida albicans, Malassezia furfur, and Malassezia globosa). In addition, minimum inhibitory concentration (MIC) of the extracts was determined by microbroth dilution method. Maximum inhibition zone of 32.00 ± 11.64 mm was exhibited when combined ethyl acetate extract of both plants was applied against M. globosa. Best effect of MIC was demonstrated by ethyl acetate extract against most tested dermatomycotic fungi. Average MIC of ethyl acetate and methanol extracts ranged as follows: (0.19 ± 0.00 to 0.65 ± 0.00 mg/mL and 0.19 ± 0.00 to 0.52 ± 0.22 mg/mL) and (0.65 ± 0.22 to 1.56 ± 0.00 mg/mL and 0.19 ± 0.00 to 0.52 ± 0.00 mg/mL), respectively. Their synergistic effect was better than the effect of individual plant leaf extract. Minimum fungicidal concentration (MFC) values varied across the fungal pathogens when extracts from both plants and their combinations were used. The findings from the current study support the traditional use of P. lanceolata and S. ovata against dermatomycotic fungal infections, which could potentially be exploited for the treatment of superficial infection in humans.


Introduction
Plants have been employed as sources of medicine in Ethiopia for thousands of years to treat diferent ailments. Studies suggest that about 80% of the Ethiopian population still depend on medicinal herbs for their basic healthcare system [1][2][3]. Even though traditional medicine and medicinal plants have a great role in the primary healthcare system in Ethiopia, the work that has been performed so far in the country to properly document and promote the associated knowledge concerning traditional medicine is scanty [4]. Terefore, documentation and promotion of traditional medicine and medicinal plants is considered necessary so that the knowledge and understanding on the extent and characteristics of traditional medicine can be preserved and utilized sustainably.
Because of their capability in providing many benefts to diferent socio-economic groups worldwide, especially in the line of modern medicine and pharmaceuticals, medicinal plants have gained great attention than ever. A wide range of plants parts such as leaves, roots, stems, bark, fowers, and seeds are used for therapeutic purposes as well as serve as precursors for the synthesis of various useful modern drugs due to their ethnomedical importance in nature. Te medicinal potentials of these plants could be traceable to the bioactive phytochemical constituents that are responsible for the physiological action on the human body. Due to their versatility and multipurpose applications, plant-derived substances have gained attention recently. Tese plantderived substances which enhance the usefulness of the plant are classifed as phytochemicals [5].
Among many popular medicinal plants, P. lanceolata and S. ovata have been reported to cure numerous diseases from cold to hepatitis, skin diseases, infectious diseases, problems concerning the digestive organs, respiratory organs, reproduction, the circulation, and for reducing fever [6].
P. lanceolata and S. ovata have been used either whole or crushed leaves, or juice from leaves of the two plants have been used to treat burns, stop bleeding, and treat all kinds of wounds to enhance the healing process [6].
As there are highly unexplored natural resources available in Ethiopia, focusing on searching for a novel, highly efcacious, better active, nontoxic, and afordable antifungal agents must be prior activity. In addition, there is scarcity of information about the synergistic antidermatomycotic activity leaf extracts of P. lanceolata and S. ovata in any part of the world including Ethiopia. Terefore, the current study aimed at evaluating the antimicrobial activity of P. lanceolata and S. ovata leaf extracts against dermatomycotic fungi that cause superfcial infection in humans. Te solvent extracts from the two plants were tested separately and synergistically.

Plant Materials Collection and Extraction.
Leaves of P. lanceolata and S. ovata plants ( Figure 1) were collected from University of Gondar compound, Northwest Ethiopia. Taxonomical identifcation and verifcation was performed at the department of biology by a Botanist in the University of Gondar.
Te collected leaves were washed repeatedly and dried in an open air and protected from direct exposure to sun light to constant weight. Air dried leaves were grounded by analytical mill and packed in plastic bags.
Ethyl acetate and methanol solvents were used for extraction of P. lanceolata and S. ovata leaves, 30 g of dried and powdered of leaves of both plants were extracted with 300 mL of 99% ethyl acetate and 80% methanol in separate fasks and were shaked for about 7 days [9], then the obtained extracts were fltered using Whatman No. 1 flter paper and the solvents were evaporated using rotary distillation apparatus. In order to obtain a complete dry extract, the resultant extracts were transferred to glass dishes and left in 40°C oven for 24 h. Te following extracts were obtained: methanolic extract (7 g) and ethyl acetate extract (11 g) from P. lanceolata leaf as well as methanolic extract (8 g) and ethyl acetate (9 g) from S. ovata leaf. Ten, they were kept at 4°C until the assessments of their antidermatomycotic activities.

Qualitative Analysis of Phytochemicals.
Te presence of diferent phytochemical constituents of P. lanceolata and S. ovata leaves was detected using standard procedures. Te presence of favonoids, alkaloids, saponins tannins, and cardiac glycosides was characterized following the method of Adesegun et al. [10]. Steroids [11] and quinones [12] were also characterized. Furthermore, the presence of phenols, terpinoids, and carbohydrates was detected using the procedure of Martin et al. [13].

Antidermatomycotic Test.
Te antidermatomycotic efect of the extracts was assessed using agar well difusion assay [14]. Te test pathogenic dermatomycotic fungi, namely, C. albicans, M. furfur, and M. globosa were obtained from microbiology unit, University of Gondar Comprehensive Specialized Hospital. Tese pathogenic isolates were frst grown on sabouraud dextrose broth, incubated for 48 h. About 20 mL of sterilized SDA (g/L: glucose 40, peptone 10, and agar 15) medium was poured into Petri dishes. Suspension of the superfcial pathogenic fungi was made in sterile normal saline and adjusted to the 0.5 MCFarland's standard. Small volume (20 µL) of fungal suspension was added to each SDA plate and then evenly distributed by means of sterile swap over the agar surface. Agar wells were prepared using a sterilized cork borer with 6 mm diameter, 2 mm deep, and about 2.5 cm apart to minimize over lapping of zone. Dimethyl sulphoxide (DMSO) was used as negative control, while ketoconazole (KC) was used as positive control.
About 100 mg of dried leaf extract powder was resuspended in 1 mL of 10% DMSO. About 50 µL of leaf extracts of both plants and ketoconazole or DMSO was placed into wells. Crude extracts and standard antibiotic were allowed to difuse for about 40 min before incubation, and then the plates were incubated in an upright position at 30°C for 5 days. Results of the qualitative screening were recorded as the average diameter of the inhibition zone surrounding the wells containing the test solution. Results were compared with ketoconazole and DMSO.
Additionally, P. lanceolata-S. ovata leaf extract mixtures were prepared by mixing 25 µL P. lanceolata leaf extract and 25 µL S. ovata leaf extract that sum up 50 µL, and similar procedures were followed for the remaining activities as it was performed for separate plant leaf extracts.

Determination of MIC and MFC.
Te MIC is the lowest concentration where no viability was observed after 24 h on the basis of metabolic activity. MIC value of each crude plant extract was estimated using broth microdilution method [15,16]. 100 μL of sabouraud dextrose broth was added to wells of a microtiter plate for each pathogenic fungal strain. A 100 μL of each dissolved crude plant leaf extract (100 mg/ mL) was then added in the frst well and serially diluted row 2 Evidence-Based Complementary and Alternative Medicine by row to give 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.19 mg/mL, and 100 μL of the mixture was discarded from the last row, thus leaving each diluted well with a volume of 100 μL, except for the positive and negative controls. Te test strains inocula were prepared from fresh 48 h grown cultures and were adjusted to McFarland standard, and 50 μL was poured to the wells except the last well. Wells containing fungal suspensions and growth media, as well as DMSO, were used as negative control, and wells having fungal suspensions and growth media, as well as KC, were used as positive control. Te wells were incubated for 72 h at 37°C temperature. Ten, 20 μL of resazurin reagent was added to each and every well to indicate respiratory activity, and a change in color from blue to pink was determined after incubating it for 72 h at 37°C temperature.
On the other hand, MFC was determined by frst selecting the plate that showed no growth during MIC determination; a loopful from each plate subcultured onto SDA was incubated for further 48 h at 37°C. Te lowest concentration at which no colonies were observed was noted as the MFC.

Data Analysis.
Te data collected through laboratory experiment were entered to Microsoft Excel 2013 and transferred to SPSS software version 20 for analysis. Te results were expressed as mean value ± standard deviation (SD) of three replicates. Where applicable, the data were subjected to ANOVA, and diferences between samples were determined by two-tailed t-test after Bonferroni error correction of the predictive value. P ≤ 0.05 was considered statistically signifcant.

Phytochemical Detection.
Te diferent photochemical components of extracts of leaves of both plants were undertaken. All the tested phytochemical components, i.e., steroids, terpenoids, phenols, saponins, tannins, alkaloids, favonoids, carbohydrates, quinines, and glycosides were found in P. lanceolata extract; however, steroids and tannins were not detected in S. ovata leaf extract.

Antidermatomycotic Fungal Activities.
Test fungal pathogens used in the current study were C. albicans, M. furfur, and M. globosa. Both ethyl acetate and methanol extracts of P. lanceolata were assayed against these pathogens using agar well difusion assay. Te result in the current study revealed that the extracts from both plants, namely, P. lanceolata and S. ovata exhibit stronger inhibitory efect on C. albicans, M. furfur, and M. globosa in comparison to ketokonazle when applied separately and synergistically. Ethyl acetate extract showed higher efects to all tested dermatomycotic fungi. Te maximum and minimum average zones of inhibition produced by ethyl acetate extract were 28.67 ± 6.69 mm and 18.17 ± 0.35 mm against M. furfur and C. albicans, respectively (Table 1). Similarly, the maximum and minimum average zones of inhibition produced by methanol extract were 21.33 ± 0.35 mm and 16.33 ± 1.40 mm against M. furfur and C. albicans, respectively (Table 1). Extracts from both solvents showed relatively substantial activities. Te result of data analysis indicated that there was a signifcant antifungal efect diference between ethyl acetate and methanol extracts against M. furfur and M. globosa. Teir efect was signifcantly diferent from ketoconazole (p ≤ 0.05) ( Table 1). Statistically, no signifcantly diferent result was seen with both extracts against C. albicans positive control (p ≥ 0.05).
Concerning S. ovata leaf extract, M. furfur and M. globosa showed variable susceptibility to ethyl acetate and methanol extracts. Tere was relatively some degree of similar susceptibility observed by C. albicans to ethyl acetate extract (15.00 ± 0.58 mm) and methanol extract (15.00 ± 0.19 mm). Te result obtained revealed that KC had better efect than both extracts. None of the extracts had better efect than KC when applied against C. albicans (21.00 ± 0.00 mm) and M. furfur (19.00 ± 0.00 mm) ( Table 2). Statistically, no signifcant diference was seen with both extracts against C. albicans and M. furfur. However, their efect was signifcantly diferent against M. globosa (p ≥ 0.05) ( Table 2).
As clearly shown in Table 3, better results were obtained from the combination of ethyl acetate and methanol crude extracts of both plants leaves against the tested microbes. M. globosa was highly susceptible to ethyl acetate extract with the average inhibition zone of 32.00 ± 11.64 mm. Statistically, no signifcance diference was seen with both extracts against all the tested superfcial pathogenic fungi (p ≥ 0.05). But their efect was signifcantly diferent from Ketoconazole (P ≤ 0.05) ( Table 4). Evidence-Based Complementary and Alternative Medicine

Determination of MIC and MFC.
Te MIC values of the tested extracts from the plant P. lanceolata leaf against tested pathogenic fungi were generally varied from pathogen to pathogen ( Te MIC values of the tested extracts from the plant S. ovata leaf against tested microbes generally varied from pathogen to pathogen. Generally, all tested fungal pathogens were inhibited by the lowest concentration of methanol extract compared to ethyl acetate extract. Terefore, methanol extract was a better choice of extract that can exert efect on tested microbes (Table 3).
Concerning the combined efects of the two plants leaves extracts; all the tested pathogens were inhibited by less concentration of ethyl acetate extracts of the two plants, i.e., 0.19 mg/mL. Te same is true for methanol extract for both M. furfur and M. globosa with the exception of C. albicans that inhibited by 0.39 mg/mL. Based on the result obtained, the MFC values of the tested extracts from the plant P. lanceolata were generally varied in the range from 0.19 to 3.12 mg/mL. Ethyl acetate and methanol were the most potent extracts with a MFC value of 0.19 mg/mL for C. albicans and M. furfur, respectively. Higher (3.12 mg/mL) MFC was recorded against C. albicans by methanol extract. Te MFC values recorded by ethyl acetate and methanol were 1.56 ± 0.00 and 0.39 ± 0.00 mg/mL against M. globosa, respectively.
Te combined extracts from both plants showed considerable synergistic efects on M. furfur. But the best result was recorded by ethyl acetate extract at a concentration of 0.19 ± 0.00 mg/mL against M. furfur and M. globosa. On the other hand, C. albicans, M. furfur, and M. globosa could be killed by methanol extract at the concentrations of 0.52 ± 0.00 mg/mL, 0.19 ± 0.00 mg/mL, and 0.39 ± 0.00 mg/ mL, respectively (Table 3). In general, varied MIC and MFC values were obtained; however, signifcantly better yields were obtained when the combined extracts act up on the dermatomycotic fungi.

Discussion
In the current study, qualitative phytochemical analysis of both P. lanceolata and S. ovata leaf extracts was undertaken. P. lanceolata leaf crude extract found to contain steroids, terpenoids, phenols, saponins, tannins, alkaloids, favonoids, carbohydrates, quinones, and glycosides. Te above fnding was in line with the work peformed by Fayera et al. [8], who confrmed secondary metabolites belonging to diferent phytochemical groups from this plant including saponins, tannins, alkaloids, terpenoids, favonoids, and phenolic compounds. Likewise, crude extracts of S. ovata leaf were found to contain terpenoids, phenols, saponins, alkaloids, favonoids, carbohydrates, quinones, and glycosides. Te above fnding was in line with the research report by Irobi et al. [17] whose analysis of this plant showed the presence of carbohydrates, alkaloids, saponins, fxed oil, and phytosterols. Te above fnding was still in concurrent with the study report carried out by Souza et al. [18] whose analysis of Spiranthera odoratissima showed the presence of organic acids, reducing sugars, favonoids, saponin compounds, coumarin compounds, phenolics, tannins, purine compounds, catechins, favonol derivatives, sesquiterpene lactones, and anthraquinones. Tis shows that the phytochemicals from the studied plants leaves extracts had contributed to their antimicrobial activity against the tested clinically isolated dermatomycotic fungi. Studies suggest that phenolic compounds disrupt microbial cell membranes [19] by changing the permeability and causing the leakage of cellular content or interfere with membrane proteins resulting in structure disrupting [20][21][22][23]. Flavonoids also afect the cell membranes of microorganisms and inhibit nucleic acid synthesis (caused by topoisomerase inhibition) and energy metabolism (caused by NADH-cytochrome c reductase or ATP synthase inhibition) [24,25]. In addition, this secondary metabolite interrupts cell wall and cell membrane synthesis. On the other hand, alkaloids possess the ability to disrupt the cell wall, inhibit DNA synthesis, and interrupt activity of enzymes (esterase, DNA-polymerase, and RNA-polymerase) or cell respiration [26]. Quinones have a potential to form irreversible complex with nucleophilic amino acids in proteins. Probable targets in the  21.00 ± 0.00 a 19.00 ± 0.00 c 15.00 ± 0.00 c DMSO 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 d Values described by the diferent letters within the same column are signifcantly diferent (P ≤ 0.05). DMSO: dimethyl sulphoxide and KC-: ketoconazole.
microbial cell are surface-exposed adhesins, cell wall polypeptides, and membrane-bound enzymes [21]. Tannins may act by inactivating/inhibiting microbial adhesins, enzymes, and cell envelope transport proteins [27]. Tannins also afect microorganisms in a number of ways which include destabilizing their cell membrane, disrupting their metabolism, as well as depriving the substrates required for microbial growth [22]. Te three tested human pathogenic dermatomycotic fungi showed diferent antimicrobial susceptibility to leaf extracts of both plants. Te possible reason for this diference in antifungal activity could be the susceptibility pattern of the pathogens and the diferences in mechanism of actions of the metabolites in each solvent fraction.
In this study, all the crude extracts were applied against three dermatomycotic (superfcial) fungi. So, the antifungal activity of ethyl acetate and methanol extracts from the plant P. lanceoleta was tested against three fungal pathogens. It was found that the plant had antifungal activity against those microbes. Tis fnding was in accordance with the studies conducted in Ethiopia by Fayera et al. [8].
Previous studies on evaluating antimicrobial activities of the two medicinal plants against dermatomycotic fungi are scanty; the results cannot be compared with any other studies. However, we have tried to compare the results against the works performed on pathogenic bacteria and fungi.
Te antimicrobial activity of P. lanceolata in this study is concurrent with the fndings of a study by Eshetie et al. [28]. Likewise, the plant S. ovata showed diferent antifungal activities to the three tested dermatomycotic fungal pathogens. Te possible explanation for this is that the plants are rich in compounds with potential antifungal activities.
Ethyl acetate extract from P. lanceolata showed higher efects to all tested fungi compared to methanol extract with the average zones of inhibition 28.67 ± 6.69 mm, 27.00 ± 1.00 mm, and 18.17 ± 0.35 mm against M. furfur, M. globosa, and C. albicans, respectively. Te average zones of inhibition formed by ethyl acetate were better than the report from Eshetie et al. [28]. Tis diference could be due to methodology diference, the age of the plant, and the difference in the defense mechanism against foreign chemicals by bacterial and fungal species.
Te current study indicated that both ethyl acetate and methanol extracts of the plant S. ovata leaf had good antifungal efect on C. albicans, M. furfur, and M. globosa. Better result was obtained from the study report by Akilandeswariet et al. [29] on antimicrobial activity of leaf extracts of S. acuta Burm related species. On the other hand, Abdulrahman et al. [30] obtained poor result on the same plant leaf extract. Te variation could be diferences in ways of extract preparation, the diference in the species of plants, the diferent geographical and environmental conditions during the growth of the plant as well as the diference in the age of the plants.
According to the fndings of this study, the synergistic efect P. lanceolata and S. ovata leaf extracts had larger inhibition zone against tested dermatomycotic fungal pathogens than that of the leave extract of each plant applied separately. Te synergistic efect may be attributed to the possibilities of the extracts afecting the diferent fungi processes in the cell [31]. Tis might be the basic reason why the local community widely uses the mixture of crude extracts from those plants to treat diferent pathogenic fungal infections.
Te present study revealed that the lowest MIC values observed by ethyl acetate extract from the P. lanceolata leaf against M. furfur and C. albicans were 0.19 ± 0.00, while the MIC value of the same extract against M. globosa was  Evidence-Based Complementary and Alternative Medicine 0.65 ± 0.00. Te MIC value recorded in this study was much better than the fnding report by Eshetie et al. [28] who reported the MIC value of 12.5 mg/mL-50 mg/mL against diferent tested bacteria. Tis diference could be due to experimental pathogens utilized. Te plant could be efective for fungal pathogens compared to bacterial pathogens. Concerning S. ovata leaf extracts, all the tested fungal pathogens were inhibited by the lowest concentration of methanol extract compared to ethyl acetate extract. Terefore, methanol extract was a better choice of extract that can exert efect on the tested microbes (Table 3).
Concerning the MIC value of combined efects of the extracts, the two extracts proven to have an increased efcacy (lowest concentration of 0.19 mg/mL) when compared to individual application of extracts (Table 3). Te same is true for methanol extract for both M. furfur and M. globosa with the exception of C. albicans that inhibited by 0.39 mg/ mL (Table 3).

Conclusion
From the overall study, it can be concluded that extracts from P. lanceolata and S. ovata leaf were found to contain various phytochemical secondary metabolites. Te extracts from both plants showed antimicrobial activity when used against the superfcial human fungal pathogens. Te combined efect of crude extracts from both plants leaves was found to have higher antifungal activity than the efect of individual plant leaf extracts. Te fndings from the current study support the traditional use of P. lanceolata and S. ovata against dermatomycotic fungal infections, which could potentially be exploited for the treatment of superfcial infection in humans.

Data Availability
Te data used in this study are included in the article.